Galuska Sebastian P, Geyer Rudolf, Gerardy-Schahn Rita, Mühlenhoff Martina, Geyer Hildegard
Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, Giessen D-35392 Germany.
Institute of Cellular Chemistry, Medical School Hannover, Hannover D-30625, Germany.
J Biol Chem. 2008 Jan 4;283(1):17-28. doi: 10.1074/jbc.M707024200. Epub 2007 Nov 6.
Polysialic acid (polySia), an alpha2,8-linked polymer of N-acetylneuraminic acid, represents an essential regulator of neural cell adhesion molecule (NCAM) functions. Two polysialyltransferases, ST8SiaII and ST8SiaIV, account for polySia synthesis, but their individual roles in vivo are still not fully understood. Previous in vitro studies defined differences between the two enzymes in their usage of the two NCAM N-glycosylation sites affected and suggested a synergistic effect. Using mutant mice, lacking either enzyme, we now assessed in vivo the contribution of ST8SiaII and ST8SiaIV to polysialylation of NCAM. PolySia-NCAM was isolated from mouse brains and trypsinized, and polysialylated glycopeptides as well as glycans were analyzed in detail. Our results revealed an identical glycosylation and almost complete polysialylation of N-glycosylation sites 5 and 6 in polySia-NCAM irrespective of the enzyme present. The same sets of glycans were substituted by identical numbers of polySia chains in vivo, the length distribution of which, however, differed with the enzyme setting. Expression of ST8SiaIV alone led to higher amounts of short polySia chains and gradual decrease with length, whereas exclusive action of ST8SiaII evoked a slight reduction in long polySia chains only. These variations were most pronounced at N-glycosylation site 5, whereas the polysialylation pattern at N-glycosylation site 6 did not differ between NCAM from wild-type and ST8SiaII- or ST8SiaIV-deficient mice. Thus, our fine structure analyses suggest a comparable quality of polysialylation by ST8SiaII and ST8SiaIV and a distinct synergistic action of the two enzymes in the synthesis of long polySia chains at N-glycosylation site 5 in vivo.
多唾液酸（polySia）是一种由N-乙酰神经氨酸通过α2,8连接而成的聚合物，是神经细胞黏附分子（NCAM）功能的重要调节因子。两种多唾液酸转移酶，即ST8SiaII和ST8SiaIV，负责polySia的合成，但其在体内的各自作用仍未完全明确。先前的体外研究确定了这两种酶在影响NCAM的两个N-糖基化位点使用上的差异，并提示存在协同效应。我们利用缺失其中一种酶的突变小鼠，在体内评估了ST8SiaII和ST8SiaIV对NCAM多唾液酸化的贡献。从鼠脑中分离出多唾液酸-NCAM并进行胰蛋白酶消化，对多唾液酸化糖肽以及聚糖进行了详细分析。我们的结果显示，无论存在哪种酶，多唾液酸-NCAM中N-糖基化位点5和6的糖基化相同且几乎完全多唾液酸化。在体内，相同的聚糖被相同数量的多唾液酸链取代，然而其长度分布因酶的不同而有所差异。单独表达ST8SiaIV会导致短多唾液酸链数量增加，且随着长度增加逐渐减少，而ST8SiaII的单独作用仅使长多唾液酸链略有减少。这些差异在N-糖基化位点5最为明显，而野生型小鼠与ST8SiaII或ST8SiaIV缺陷型小鼠的NCAM在N-糖基化位点6的多唾液酸化模式并无差异。因此，我们的精细结构分析表明，ST8SiaII和ST8SiaIV的多唾液酸化质量相当，且在体内N-糖基化位点5合成长多唾液酸链时，这两种酶具有独特的协同作用。
